Development of low molecular weight HIV fusion inhibitors based on a carboxybenzyl-substituted indole scaffold is proposed. Early lead compounds show promising binding affinity to a deep hydrophobic cavity on the coiled coil of HIV-1 gp41, correlated with inhibitory activity against cell-cell and viral cell fusion. Successful inhibition of gp41 provides a universal barrier to initial infection by HIV, and has been shown to have important immune-protective effects in an infected individual by preventing the spread of infection to bystander cells. Currently, fusion inhibitors are limited to peptides derived from the gp41 C-heptad repeat, which, while highly efficacious, have pharmacological limitations such as route of administration, half-life and cost. Small molecule inhibition of HIV fusion has been a long sought after but elusive goal and success in this area would mark a significant improvement in available therapies as well as potential for prophylaxis in a microbicidal formulation. We plan synthesis of 50 - 100 new compounds to decipher the pharmacophore required to inhibit the protein - protein interaction involved in the gp41 N - C-peptide interaction, relying on relatively simple chemistry to make modifications to the basic scaffold. We have specific biochemical assays to determine affinity of our compounds for the known hydrophobic cavity in gp41, and will correlate binding activity with biological activity using cell-cell and viral cell fusion assays in the presence or absence of additives such as serum, seminal and cervical fluid. We have also developed the foundation for a novel NMR method for structure determination of bound ligands, which will be expanded for use with the high affinity ligands being developed in this proposal. From a starting compound with 55M binding affinity and 95M IC50 for fusion inhibition, we have synthesized several next generation inhibitors, and have obtained a 10-fold improvement in activity. Our goal is to obtain inhibitors that are at least 100-fold improved over the lead, having < 100nM IC50 for fusion inhibition. Thus our specific aims are 1) to utilize our current understanding of structure-activity relationships to design a new set of indole containing inhibitors, and to use binding, cell-cell fusion and viral-cell fusion assays to confirm activity, mechanism and specificity for HIV; 2) to develop NMR procedures to obtain experimental restraints for computational structure determination of bound ligands, as an aid to the rational design of improved inhibitors. Construction and testing of this molecular library promises to result in low molecular weight inhibitors with greatly improved biological activity against HIV fusion and with a well-defined mechanism of action. Such compounds could be developed into inexpensive non-peptide fusion inhibitors, which would revolutionize both treatment options and prophylaxis for the millions of new infections that occur worldwide annually. PUBLIC HEALTH RELEVANCE: The prevalence of HIV infection remains a significant public health problem due to the development of viral strains resistant to current treatments, and due to spread of the virus from individuals who are unaware they are infected. This proposal seeks to apply structure-aided drug design to develop low molecular weight HIV fusion inhibitors which prevent viral entry and the cell-to-cell spread that causes immune deficiency. Drugs in this class would be useful in microbicides to prevent infection, or could be given orally to treat infection as an alternative to the currently used fusion inhibitor Enfuvirtide(R), which must be given intravenously.